Therapy-enhancing glucan

ABSTRACT

This invention provides a method for introducing substances into cells comprising contacting a composition comprising orally administered beta-glucan with said cells. This invention also provides a method for introducing substances into a subject comprising administering to the subject an effective amount of the above compositions. The substance. which could be delivered orally includes but is not limited to peptides, proteins, RNAs, DNAs, chemotherapeutic agents, biologically active agents, plasmids, and other small molecules and compounds. Finally, this invention provides a composition comprising orally administered beta-glucan capable of enhancing efficacy of IgM and different uses of the said composition.

This application is a continuation-in-part of U.S. Ser. No. 10/621,027,Filed Jul. 16, 2003, the contents of which are incorporated in itsentirety by reference here into this application.

Throughout this application, various references are cited. Disclosuresof these publications in their entireties are hereby incorporated byreference into this application to more fully describe the state of theart to which this invention pertains.

BACKGROUND OF THE INVENTION

This disclosure relates to a method for introducing substances intocells comprising contacting a composition comprising orally administeredbeta-glucan with said cells. A feature of this invention provides amethod for introducing substances into a subject comprisingadministering to the subject an effective amount of the abovecompositions. The substance which could be delivered orally includes butis not limited to peptides, proteins, RNAs, DNAs, chemotherapeuticagents, biologically active agents, and plasmids. Other small moleculesand compounds may be used as well. Another feature of the presentinvention is a composition comprising orally administered beta-glucancapable of enhancing efficacy of IgM antibodies.

Glucans derived from cell walls of yeasts, such as Saccharomycescervisiae or mutant yeast strains described in U.S. Pat. No. 5,250,436,the disclosure of which is incorporated herein in its entirety byreference, may be used in the above compositions. Glucans having β(1-3)and β(1-6) linkages may be prepared by the process described in U.S.Pat. Nos. 5,233,491 and 4,810,646, the disclosures of which areincorporated herein in their entirety by reference. Soluble or aqueousglucans which are suitable for oral administration may be produced bythe process described in U.S. Pat. Nos. 4,810,646 and 5,519,009, thedisclosures of which are incorporated herein in their entirety byreference.

Beta-glucans have been tested for tumor therapy in mice for nearly 40years.^(1,2) Several forms of mushroom derived beta-glucans are usedclinically to treat cancer in Japan, including PSK (from Coriolusversicolor), Lentinan and Schizophyllan. In randomized trials in Japan,PSK has moderately, but significantly improved survival rates in somecancer trials: after gastrectomy,^(3,4) colorectal surgery,^(5,6) andesophagectomy⁷ to remove primary tumors. Results have been lessencouraging in breast cancer,^(8,9) and leukemia.¹⁰ Schizophyllan hasimproved survival of patients with operable gastric cancer,¹¹ inoperablegastric cancer,^(12,13) and cervical cancer.¹⁴ Again, though survivaldifferences between groups were statistically significant, theseimprovements were modest. While beta-glucans are not widely used byWestern oncologists, beta-glucan containing botanical medicines such asReishi and maitake¹⁵ are widely used by U.S. cancer patients asalternative/complementary cancer therapies. These previous studies thatlooked for a therapeutic effect of beta-glucan, did not incorporateco-administration of therapeutic monoclonal antibodies (MoAb) as part ofthe protocol. There is increasing evidence that antibody is necessary todeposit iC3b which acts as a potent opsonin of human tumors. Whenbeta-glucan is administered without co-administration of MoAb, its tumorcytotoxic effect requires the presence of naturally-occurring antitumorantibodies which can be quite variable among patients and even inexperimental mice.

Anti-tumor effect of beta-glucan when combined with cancer specificantibodies was previously described. Previous studies have shown thatoral beta-glucans derived from barley or oats can greatly enhance theanti-tumor activity of anti-tumor monoclonal antibodies in xenographmodels. See Therapy-Enhancing Glucan, Int'l Application No.PCT/US02/01276, filed Jan. 15, 2002. Cheung et al., Oral(1-3),(1-4)-beta-glucan syngergizes with anti-ganglioside GD2 monoclonalantibody 3F8 in the therapy of neuroblastoma. Clin Cancer Res.2002;8:1217-1223. Cheung N K et al., Orally administered beta-glucansenhance anti-tumor effects of monoclonal antibodies. Cancer ImmunolImmunother. 2002; 51:557-564. The phase I clinical trial supports theprediction that barley beta-glucan can enhance the antibody effect onmetastatic cancer. As previously noted, lentinan and laminarin, both(1→3), (1→6)-β-D-glucans, were not as effective as barley glucan.¹⁶ Inaddition, among the (1→3), (1→4)-β-D-glucans, small molecular weightpreparations and Lichenans were not as effective. The molecular size andthe fine structure of beta-glucan may have substantial influence ontheir synergistic effect on antibodies towards tumors.

In Europe and USA beta-glucans especially from Bakers' yeast have longbeen employed as feed additives for animals, as dietary supplement forhumans,¹⁷ in treatment of wounds,¹⁸ and as an active ingredient in skincream formulations. The basic structural unit in beta-glucans is theβ(1→3)-linked glucosyl units. Depending upon the source and method ofisolation, beta-glucans have various degrees of branching and oflinkages in the side chains. The frequency and hinge-structure of sidechains determines its immunomodulor effect. beta-glucans of fungal andyeast origin are normally insoluble in water, but can be made solubleeither by acid hydrolysis or by derivatisation introducing chargedgroups like -phosphate, -sulphate, -amine, -carboxymethyl and so forthto the molecule.^(19,20)

Soluble glucan with the molecular structure where (1→3)-β-D-glucan unitsform the backbone with branches made up of (1→3)-β-D-glucan unitspositioned at (1→6)-β-D-glucan hinges was isolated from Baker's yeast,Saccharomyces cerevisiae. High molecular weight fractions were obtainedand

tested for synergy with monoclonal antibodies in tumor models. Theanti-tumor effect of soluble yeast beta-glucan was found to becomparable to the anti-tumor effect of soluble barley beta-glucan, whencombined with monoclonal antibodies specific for human cancer asdetailed below.

SUMMARY OF THE INVENTION

This invention provides a method for introducing substances into cellscomprising contacting a composition comprising orally administeredbeta-glucan with said cells.

Another aspect of the present is a method for introducing substancesinto a subject comprising administering to the subject an effectiveamount of the above compositions. The substance which could be deliveredorally includes but is not limited to peptides, proteins, RNAs, DNAs,chemotherapeutic agents, biologically active agents, and plasmids. Othersmall molecules and compounds may be used as well.

A further aspect of the present invention is a composition comprisingorally administered beta-glucan capable of enhancing efficacy of IgMantibodies.

DETAILED DESCRIPTION OF THE FIGURES

FIG. 1. Barley (1→3), (1→4)-β-D-glucan plus antibody in the treatment ofmetastatic neuroblastoma in patients. MIBG scan before and aftertreatment in a patient with metastatic neuroblastoma refractory tomultiple regimens of chemotherapy. Patient received intravenous anti-GD2antibody 3F8 (10 mg/m2/day) for a total of 10 days, plus oral barleybeta-glucan over the same time period. FIG. 1A shows baseline MIBG scanof patient. Extensive osseous metastasis can be seen in the femora,fibulae, pelvis, ribs, left scapula, right clavicle, humeri, skull andspine. Heart, liver, stomach and colon uptakes are physiologic. FIG. 1Bshows MIBG scan of same patient 2 months later, following a single cycleof therapy with 3F8 plus glucan. Areas of metastases have significantlyimproved.

FIG. 2. Barley (1→3), (1→4)-β-D-glucan plus antibody in treatment ofsubcutaneous human lymphoma xenografted in SCID mice. SCID mice withestablished subcutaneous Daudi (n=9) (FIG. 2A), Hs445 (n=5) (FIG. 2B),EBV-derived LCL (n=9) (FIG. 2C) and RPMI 6666 (n=10; data not shown)xenografts were treated either with 200ug intravenous rituximab twiceweekly for 8 doses (▪), 400 ug (1→3), (1→4)-D-β-glucan administeredorally via intragastric gavage daily for 29 days (Δ) or a combination ofrituximab and (1→3), (1→4)-D-β-glucan (x), or left untreated (♦).Percentage tumor growth is plotted on y-axis and days after treatmentwas commenced on x-axis. Error bars represent SEM and have been shownonly for rituximab alone and combination groups. For all xenografts,only combination treatment was associated with reduction in tumorgrowth. The reduction in tumor growth per day in the group receivingbeta-glucan in addition to rituximab compared to rituximab alone was2.0% (95% CI 1.3-2.7%; p<0.0005) for Daudi, 0.8% for EBV-derived LCL(95% CI 0.4-1.2%; p<=001), 2.2% for Hs445 (95% C.I. 1.2%-3.2%;p=0.0009), and 1.8% for RPMI6666 (95% CI 1.0-2.7%; p<0.0002; data notshown) xenografts.

FIG. 3. Barley (1→3),(1→4)-β-D-glucan plus antibody in treatment ofdisseminated human lymphoma xenografted in SCID mice. 5×10⁶ Daudi (FIG.3A) or Hs445 (FIG. 3B) cells in 100 μl normal saline were injectedintravenously (IV) into SCID mice. Mice were treated either with 200 ugintravenous rituximab twice weekly for 8 doses ( . . . ), 400 ug (1→3),(1→4)-D-β-glucan administered orally via intragastric gavage daily for29 days ( . . . ) or a combination of rituximab and (1→3),(1→4)-D-β-glucan (—), or left untreated (—) commencing 10 days aftertumor implantation. Tumors grew systemically and mice became paralyzedwhen tumor cells infiltrated the spinal canal, resulting in hind-legparalysis. Mice were sacrificed at onset of paralysis or when animalslost 10% of their body weight. Kaplan-Maier survival curves for thevarious groups are shown in FIGS. 2A (Daudi) and 2B (Hs445). Micetreated with a combination of (1→3), (1→4)-D-β-glucan and rituximab hada significantly increased survival when compared to all other treatmentgroups (p<0.0005 for Daudi and p=0.001 for Hs445) or when compared torituximab alone (p<0.0005 for Daudi and p=0.01 for Hs445). Mediansurvival for mice with no treatment, rituximab alone, BG, andrituximab+BG groups was 27,71,43 and 124 days respectively for Daudixenografts, and 12, 16, 31 and 243 days respectively for Hs445xenografts.

FIG. 4. Dose response of 3G6 (anti-GD2 IgM antibody) in the presence ofbarley β-glucan in the treatment of human neuroblastoma. Two millionLAN1 neuroblastoma cells were xenografted subcutaneously in athymicBalb/c mice. Treatment started in groups of5 mice each, 2 weeks aftertumor implantation when visible tumors reached 0.7-0.8 cm diameter. 3G6group (solid squares) was treated with 200 ug of intravenous 3G6injected through the retroorbital plexus twice weekly (M and Th). 3G6+BGgroup was treated with 200 ug i.v. 3G6 twice weekly plus oralbeta-glucan (BG) 400 ug daily by gavage for a total of 14-18 days. 3G6was administered in 3 different doses (open triangle 8 ug per dose, opensquare 40 ug, open circle 200 ug). BG group (solid circles) received 400ug oral beta-glucan alone. Tumor size was measured from the first day oftreatment, and the product of the largest diameters expressed as percentof the size on day 0 of treatment. Vertical bars represent standarderrors, depicted in only 4 groups for clarity. While BG alone and 3G6alone showed no anti-tumor effect, the BG+200 ug 3G6 group showed highlysignificant tumor shrinkage and suppression which was 3G6 dose-dependent(p<0.05).

FIG. 5. Treatment of human neuroblastoma using 3G6 (anti-GD2 IgMantibody) in the presence of yeast (1→3), (1→6)-β-D-glucan. Two millionLAN1 neuroblastoma cells were xenografted subcutaneously in athymicBalb/c mice. Treatment started in groups of 5 mice each, 2 weeks aftertumor implantation when visible tumors reached 0.7-0.8 cm diameter. 3G6group (solid squares) was treated with 200 ug of intravenous 3G6injected through the retroorbital plexus twice weekly (M and Th) for atotal of 5 doses. Particulate yeast glucan group (solid triangles)received 400 ug oral particulate yeast glucan alone. 3G6+whole yeastparticles (open diamond) was treated with 200 ug iv 3G6 twice weeklyplus yeast particles 400 ug daily by gavage for a total of 14-18 days.3G6+soluble yeast glucan group was treated with 200 ug iv 3G6 twiceweekly plus soluble yeast glucan 400 ug daily by gavage for a total of14-18 days. 3G6+particulate yeast glucan group was treated with 200 ugi.v. 3G6 twice weekly plus particulate yeast glucan 400 ug daily bygavage for a total of 14-18 days. Tumor size was measured from the firstday of treatment, and the product of the largest diameters expressed aspercent of the size on day 0 of treatment. Vertical bars representstandard errors, depicted in only 4 groups for clarity. While glucanalone and 3G6 alone showed no anti-tumor effect, soluble and particulateyeast glucan when combined with 3G6 group showed highly significanttumor shrinkage and suppression (p<0.05).

FIG. 6. Treatment of human neuroblastoma using 3F8 (anti-GD2 IgGantibody) in the presence of barley and yeast β-glucan. Two million LAN1neuroblastoma cells were xenografted subcutaneously in athymic Balb/cmice. Treatment started in groups of 5 mice each, 2 weeks after tumorimplantation when visible tumors reached 0.7-0.8 cm diameter. 3F8 group(solid diamonds) was treated with 200 ug of intravenous 3F8 injectedthrough the retroorbital plexus twice weekly (M and Th) for a total of 5doses. Barley glucan group (solid squares) received 400 ug barely glucanalone. 3F8+barley glucan group (open diamond) was treated with 200 ugi.v. 3F8 twice weekly plus barely glucan 400 ug daily by gavage for atotal of 14-18 days. 3F8+soluble yeast glucan group (open squares) wastreated with 200 ug iv 3F8 twice weekly plus soluble yeast glucan 400 ugdaily by gavage for a total of 14-18 days. Tumor size was measured fromthe first day of treatment, and the product of the largest diametersexpressed as percent of the size on day 0 of treatment. Vertical barsrepresent standard errors. While glucan alone and 3F8 alone showed noanti-tumor effect, barley and soluble yeast glucan when combined with3F8 group showed highly significant tumor shrinkage and suppression(p<0.05).

FIG. 7. Treatment of disseminating human lymphoma in SCID mice usingRituxan and barley or yeast β-glucan. 5×10e6 Daudi cells in 100 μlnormal saline were injected intravenously (IV) into SCID mice. Tumorsgrew systemically and mice became paralyzed when tumor cells infiltratedthe spinal canal, resulting in hind-leg paralysis. Mice were sacrificedat onset of paralysis or when animals lost 10% of their body weight.Therapy was initiated ten days after injection of tumor cells. 40 μgrituximab (Genentech, San Francisco, Calif.) was injected intravenouslytwice weekly for a total of eight injections and 400 μg glucanadministered orally via intragastric gavage daily for 29 days. Mice wereweighed weekly and observed clinically at least once daily. Micereceiving rituxan plus barley glucan or rituxan plus yeast solubleglucan have a highly significant prolonged survival (p<0.05).

FIG. 8 illustrates the peGP-C1 vector purchased from BD Biosciences(Palo Alto, Calif.).

FIG. 9 shows glucan facilitates gene transfer into monocytes.

FIG. 10 illustrates higher molecular weight β-glucan and gene transfer.

FIG. 11 illustrates presence of GFP mRNA in circulating monocytes.

DETAILED DESCRIPTION OF THE INVENTION

This invention provides a composition for oral uptake of substancecomprising an appropriate amount of carbohydrates. In an embodiment, thecarbohydrate is glucan.

When administered orally, glucan is taken up by macrophages andmonocytes which carry these carbohydrates to the marrow andreticuloendothelial system from where they are released, in anappropriately processed form, onto myeloid cells including neutrophils,and onto lymphoid cells including natural killer (NK) cells. Thisprocessed glucan binds to CR3 on these neutrophils and NK cells,activating them in tumor cytotoxicity in the presence of tumor-specificantibodies.

Since macrophage and monocytes ingest glucan (whether soluble, gel orparticle) from the gut, glucan is a potential conduit for gene therapy.Unlike proteins, DNA or plasmids are relatively heat-stable, and can beeasily incorporated into warm soluble barley glucan which gels whencooled to room or body temperature. When mice are fed these DNA-glucancomplexes, reporter genes can be detected in peripheral blood monocytesand macrophages within days. More importantly these reporter genes areexpressed in these cells, a few days after ingestion of these DNAcomplexes. These findings have potential biologic implications. Glucanand similar carbohydrates may be conduits for DNA or plasmids to getinto the human body. Oral glucan may be a convenient vehicle forcorrecting genetic defects of macrophages/monocytes, or administeringgenetic vaccines.

As it can easily be appreciated by an ordinary skilled artisan, othercarbohydrates capable of functioning like glucan could be identified andused in a similar fashion. One easy screening for such carbohydrates canbe established using glucan as the positive control.

The glucan includes but is not limited to β(1-3) and β(1-4) mixedlinkage-glucan, and the glucan is of high molecular weight. The glucanmay also have β(1-3) and β(1-6) linkages.

This invention also provides a method for introducing substance intocells comprising contacting the above compositions with said cells. Onecan use reporter genes or other markers to assess the efficiency of thesaid introduction. Reporter genes or markers are well known in themolecular biology field. In addition, this invention provides a methodfor introducing substance into a subject comprising administering to thesubject an effective amount of the above compositions.

This invention provides a composition for the oral delivery of one ormore substances comprising an effective amount of an orally administeredbeta-glucan and one or more chemotherapeutic agents.

In an embodiment, the glucan contains 1,3-1,6 or 1,3-1,4 mixed linkages,or a mixture of 1,3-1,6 and 1,3-1,4 mixed linkages. In anotherembodiment, the glucan enhances the efficacy of chemotherapeutic agentsor anti-cancer antibodies.

In a further embodiment, the glucan is derived from grass, plants,mushroom, yeast, barley, fungi, wheat or seaweed. The glucan may be ofhigh molecular weight. The molecular weight of the glucan may be atleast 10,000 Daltons.

In a further embodiment, the substance is a peptide, protein, RNA, DNA,plasmid, or chemotherapeutic agent. As used herein, chemotherapeuticagents include chemicals that combat disease in the body of an animal ormedications used to treat various forms of cancer.

This invention provides a method for introducing substance into cellscomprising contacting the above-described composition with said cells.

The substance which could be delivered orally includes but is notlimited to peptides, proteins, RNAs, DNAs, and plasmids. Other smallmolecules and compounds may be used as well.

This invention provides a method for treating a subject comprisingadministering to the subject an effective amount of the abovecomposition. In an embodiment, the method further comprises thesubstance.

This invention provides a method for treating a subject with geneticdisorder comprising administering to the subject an effective amount ofthe above-described composition and a substance capable of correctingsaid genetic disorder. The substance includes but is not limited to apeptide, protein, RNA, DNA, plasmid and other small molecule andcompound.

This invention provides a composition comprising an effective amount oforally administered (1→3), (1→6) beta-glucan capable of enhancingefficacy of IgM antibodies.

This invention provides a composition comprising an effective amount oforally administered (1→3), (1→6) beta-glucan capable of enhancingefficacy of antibodies. Glucans derived from cell walls of yeasts, suchas Saccharomyces cervisiae or mutant yeast strains described in U.S.Pat. No. 5,250,436, the disclosure of which is incorporated herein inits entirety by reference, may be used in the above compositions.Glucans having β(1-3) and β(1-6) linkages may be prepared by the processdescribed in U.S. Pat. Nos. 5,233,491 and4,810,646, the disclosures ofwhich are incorporated herein in their entirety by reference. Soluble oraqueous glucans which are suitable for oral administration may beproduced by the process described in U.S. Pat. Nos. 4,810,646 and5,519,009, the disclosures of which are incorporated herein in theirentirety by reference.

In an embodiment, the antibody is a monoclonal antibody, or an antibodyagainst cancer or tumor cells, which include but are not limited toanti-CEA antibody, anti-CD20 antibodies, anti-CD25 antibodies, anti-CD22antibodies, anti-HER2 antibodies, anti-tenascin antibodies, MoAb M195,Dacluzimab, anti-TAG-72 antibodies, R24, Herceptin, Rituximab, 528, IgG,IgM, IgA, C225, Epratuzumab, and MoAb 3F8. In another embodiment, theantibody is a tumor-binding antibody.

Moreover, the antibody is capable of activating complement and/oractivating the antibody dependent cell-mediated cytotoxicity. In anotherembodiment, the antibody modulates T-cell or B-cell function.

In a further embodiment, the antibody is directed at the epidermalgrowth factor receptor, a ganglioside, such as GD3 or GD2.

In a further embodiment, the antibodies are effective against cancerswhich include neuroblastoma, melanoma, non-Hodgkin's lymphoma,Epstein-Barr related lymphoma, Hodgkin's lymphoma, retinoblastoma, smallcell lung cancer, brain tumors, leukemia, epidermoid carcinoma, prostatecancer, renal cell carcinoma, transitional cell carcinoma, breastcancer, ovarian cancer, lung cancer, colon cancer, liver cancer, stomachcancer, or other gastrointestinal cancers.

In a further embodiment, the above-described composition is in apharmaceutically acceptable carrier.

This invention provides a method for treating a subject comprisingadministrating the above-described composition to a subject.

This invention provides a composition comprising an effective amount oforally administered (1→3), (1→6) beta-glucan capable of enhancingefficacy of vaccines. In an embodiment, the vaccine is against cancer orinfectious agents, such as bacteria, viruses, fungi, or parasites.

This invention provides a composition comprising an effective amount oforally administered (1→3), (1→6) beta-glucan capable of enhancingefficacy of natural antibodies or infectious agents.

This invention provides a composition comprising an effective amount oforally administered (1→3), (1→6) beta-glucan capable of enhancing hostimmunity.

This invention provides a composition comprising an effective amount oforally administered (1→3), (1→6) beta-glucan capable of enhancing theaction of an agent in preventing tissue rejection. In an embodiment, thetissue is transplanted tissue or transplanted organ or the host as ingraft-versus-host disease.

In an embodiment, the glucan of the above-described composition has highmolecular weight. The molecular weight of glucan is at least 10,000Daltons. In another embodiment, the glucan is derived from barley, oat,mushroom, seaweed, fungi, yeast, wheat or moss. In a further embodiment,the glucan is stable to heat treatment.

In a further embodiment, above-describe composition is stable afterboiling for 3 hours. The effective dose of the above-describedcomposition is about >=25 mg/kg/day, five days a week for a total of 2-4weeks.

The invention will be better understood by reference to the ExperimentalDetails which follow, but those skilled in the art will readilyappreciate that the specific experiments detailed are only illustrative,and are not meant to limit the invention as described herein, which isdefined by the claims which follow thereafter.

EXAMPLE I

Phase I Study of Barley β-Glucan in Combination with Anti-GD2 Antibodyin Stage 4 Neuroblastoma.

A total of 24 patients were studied. These patients are all children oradolescents with relapsed or refractory stage 4neuroblastoma metastaticto bone, marrow or distant lymph nodes, some with large soft tissuemasses. Beta-glucan was well tolerated with no dose-limiting toxicities.Anti-tumor responses were recorded for marrow disease (histology, MIBGscans), soft tissue tumors (CT), as well biochemical markers (urine VMAand HVA tumor markers). One example of tumor response is shown in FIGS.1A and 1B: ¹³¹I-metaiodobenzylguanidine (MIBG) scans showingnear-complete resolution of extensive metastases after one treatmentcycle of 3F8 plus beta-glucan. These responses are uncommon in patientswith refractory or relapsed metastatic stage 4 NB treated with 3F8 aloneor 3F8 in combination with cytokines. The best response rate for 3F8 todate was in a Phase II trial of combination 3F8 plus GMCSF where 7 of 33(21%) children achieved MIBG improvement. In contrast, 62% (13 of 21)evaluable patients on 3F8+beta-glucan had MIBG improvement, a neartripling of the response rate (p=0.008 by χ²). In addition, among 15patients with marrow disease, 5 achieved complete marrow remission(30%), and 8 with stable disease in the marrow. (See FIG. 1)

EXAMPLE II

Rituximab activates complement-mediated and antibody-dependentcell-mediated cytotoxicities, and is effective against B-cell lymphomas.Beta-glucans are naturally occurring glucose polymers that bind to thelectin domain of CR3, a receptor widely expressed among leukocytes,priming it for binding to iC3b activated by antibodies. Barley-derived(1→3), (1→4)-β-D-glucan (BG), when administered orally (400 μg perday×29 days), strongly synergized with subtherapeutic doses ofintravenous rituximab (200 μg twice/week×8 doses) in the therapy ofCD20-positive human lymphomas. Growth of established subcutaneousnon-Hodgkin's lymphoma (NHL) (Daudi and EBV-derived B-NHL) or Hodgkin'sdisease (Hs445 or RPM16666) xenografted in SCID mice was significantlysuppressed, when compared to mice treated with rituximab or BG alone.Survival of mice with disseminated lymphoma (Daudi and Hs445) wassignificantly increased. There was no weight loss or clinical toxicityin treated animals. This therapeutic efficacy and lack of toxicity of BGplus rituximab supports further investigation into its clinical utility.

Introduction

The chimeric anti-CD20 antibody rituximab is being evaluated in anincreasing number of disorders. After clinical efficacy was initiallydemonstrated against relapsed and refractory follicular/low gradenon-Hodgkin's lymphomal, responses to rituximab have been reported inother malignant and non-malignant B-cell disorders². Several mechanismsof action have been proposed including activation of apoptoticpathways³, elaboration of cytokines⁴, and elicitation of hostcomplement-dependent cytotoxicity (CDC) and antibody-dependentcell-mediated cytotoxicity (ADCC)⁵. Although many patients with B-celldisorders respond to rituximab, remissions are often transient⁶. Morethan 50% of lymphomas recurrent after rituximab treatment failed torespond the second time⁷. Mechanisms of resistance to rituximab are asyet unclear, and may include paucity or loss of target antigen⁸,pharmacokinetic variations among individual patients, FcR polymorphism⁹,resistance to complement activity¹⁰, or inherent gene expression of thelymphoma¹¹. beta-glucans are complex polymers of glucose with affinityfor the lectin site of the CR3 receptor on leucocytes². With boundbeta-glucan, CR3 (CD11b) is primed to engage iC3b fragments deposited oncells by complement-activating antibodies. This receptor mediates thediapedesis of leukocytes through the endothelium and stimulatesphagocytosis, degranulation and tumor cytotoxicity. Many fungi presentbeta-glucan or beta-glucan-like CR3 binding ligands on their cellsurface. Hence, when iC3b deposition occurs, both CD11b and lectin sitesbecome engaged, and phagocytosis and respiratory burst is triggered¹³.In contrast, tumor cells lack such molecules, and even when coated withiC3b do not generally activate CR3 and cannot activate leucocytes.Soluble forms of beta-glucan bind to lectin sites and prime bothphagocytic and NK cells to kill iC3b-coated tumor targets¹⁴.

(1→3), (1→4)-D-β-glucan (BG), a soluble, barley-derived beta-glucan hasadvantages over previously studied (1→3), (1→6)-β-glucans, particularlyefficacy when administered orally and a good safety profile¹⁵. In vivosynergism between BG and the complement-fixing antibody 3F8 againsthuman neuroblastoma xenografts^(15,16) was recently demonstrated. Thesynergism between BG and rituximab against lymphoma is now reported.

Study Design

Cell Lines:

Human Burkitt's lymphoma cell line, Daudi, and Hodgkin's disease (HD)cell lines Hs445 and RPMI 6666 were purchased from American Type CultureCollection (Rockville, Md.). Human EBV-BLCL were established usingpreviously described methods¹⁷.

Mice:

Fox Chase ICR SCID mice (Taconic, White Plains, N.Y.) were maintainedunder institutionally approved guidelines and protocols.

Tumor Models:

Subcutaneous tumors were established by injecting 5×10⁶ cells suspendedin 0.1 ml of Matrigel (Becton-Dickinson, Franklin Lakes, N.J.) into miceflanks. Tumor dimensions were measured two to three times a week andtumor size was calculated as the product of the two largest diameters.Mice were sacrificed when maximum tumor dimension exceeded 20 mm. Adisseminated tumor model was established in SCID mice as previouslydescribed¹⁸. Briefly, 5×10⁶ Daudi or Hs445 cells in 100 μl normal salinewere injected intravenously into SCID mice. Tumors grew systemically andmice became paralyzed when tumor cells infiltrated the spinal cord,resulting in hind-leg paralysis. Mice were sacrificed at onset ofparalysis or when animals lost 10% of their body weight.

Treatment Regimens:

For mice with subcutaneous tumors, therapy was initiated after tumorswere established (7-8 mm diameter). For the disseminated tumor model,therapy was initiated ten days after injection of tumor cells. Groups ofat least five mice per treatment regimen received either rituximab, BG,neither or both. 200 μg rituximab (Genentech, San Francisco, Calif.) wasinjected intravenously twice weekly for a total of eight injections and400 μg BG (Sigma, St. Louis, Mo.) administered orally via intragastricgavage daily for 29 days. Animals were weighed weekly and observedclinically at least once daily.

Statistical Analysis:

Tumor growth was calculated by fitting a regression slope for eachindividual mouse to log transformed values of tumor size. Slopes werecompared between groups using t-tests using a previously describedmethod for censored observations¹⁹. Survival in mice with disseminateddisease was compared using Kaplan-Meier analysis and proportion ofdeaths was compared by Fisher's exact χ2 test. Analyses were conductedusing STATA 7

Results and Discussion

In all subcutaneous xenograft models, significant reduction in tumorgrowth was noted in mice treated with a combination of rituximab and BG.Mice treated with rituximab alone showed a modest reduction in tumorgrowth, while those treated with BG alone or left untreated had unabatedtumor growth (FIGS. 1A, 1B, 1C). All tumors except for those treatedwith combination therapy grew beyond 20 mm size and mice had to besacrificed. Mice on combination treatment had persistent tumorsuppression even after treatment was stopped. In a multivariable linearmodel of tumor growth rate, using dummy variables for treatment, theinteraction between BG and rituximab was positive and significant,demonstrating synergism.

For disseminated xenografts, there was a significant difference insurvival between the combination and control groups for both NHL and HDmodels (p<0.005, by log-rank) (FIG. 2). 5/38 mice and 2/8 mice withdisseminated Daudi and Hs445 tumors respectively treated withcombination BG and rituximab were surviving >12 months after therapy wasdiscontinued suggesting complete eradication of disease. In contrast,0/29 and 0/8 mice receiving rituximab alone in respective groupssurvived (15% vs. 0% survival; χ2=0.01). There was no significant weightloss or other clinically apparent adverse effects. That BG is absorbedcan be inferred from the fact that it could be detected intracellularlywithin fixed and permeabilized peripheral blood leucocytes byimmunofluorescence (data not shown).

In these studies, synergism between BG and rituximab was highlysignificant irrespective of the type of CD20-positive lymphoma. Improvedresponses in Daudi xenografts as compared to Hs445 may be attributableto higher CD20 expression in the former (Mean geometric fluorescencechannel for Daudi 241 compared to 184 for Hs445). When tumors thatprogressed were examined for CD20 expression by immunofluorescencestudies of single cell suspensions or indirect immunohistochemistry offrozen sections, no significant difference was noted between groupstreated with rituximab, BG alone or rituximab+BG (data not shown),indicating that treatment with rituximab+BG was not associated with lossof CD20.

Synergism between other complement-activating monoclonal antibodies andBG^(15,16) were previously demonstrated. The current data extend thisobservation to rituximab. CDC is considered an important mechanism forrituximab cytotoxicity. Rodent complement is not inhibited efficientlyby human complement regulatory proteins (mCRP). Therefore CDC can be aneffective anti-tumor mechanism in xenograft models. However in a study,at sub-therapeutic doses of antibody, rituximab-mediated ADCC and CDCwere not sufficient to effect tumor cell killing. Since BG has no directeffect on ADCC²⁰, this synergy is most likely a result of iC3b-mediatedtumor cytotoxicity. Lymphoma cells express mCRP including CD46, CD55,and CD59^(10,21). However, iC3b-mediated cytotoxicity is unaffected bythe presence of CD59 which affects only MAC-mediated complementcytotoxicity²². Furthermore, in human breast carcinoma tumors,deposition of iC3b has been demonstrated despite the presence of mCRP²³indicating that unlike their inhibitory effect on MAC, effect oniC3b-mediated tumor cytotoxicity is not absolute.

If this synergistic effect can be safely reproduced in humans,iC3b-mediated cytotoxicity may be a potential strategy to overcomerituximab resistance in patients with B-cell malignancies. Since neitherT nor B cells are required for this synergistic effect, BG may have apotential role even in immunocompromised lymphoma patients. Furthermore,in patients with autoimmune disorders, B-cell depletion may be enhancedwith this non-toxic oral therapy. Conversely, beta-glucans can enhancerelease of cytokines such as TNF-α and IL-6²⁴, and because the acutetoxicities of rituximab are also related to cytokine release secondaryto complement activation²⁵, there is a potential of increased toxicitywhen BG and rituximab are used in combination. Carefully designed phaseI studies are necessary in order to define the safety and efficacy indeveloping BG as an adjunct to rituximab therapy in the treatment ofB-cell disorders and in antibody-based therapies of other cancers.

REFERENCES FOR EXAMPLE II

1. Maloney D G, Liles T M, Czerwinski D K, Waldichuk C, Rosenberg J.Grillo-Lopez A, Levy R. Phase I clinical trial using escalatingsingle-dose infusion of chimeric anti-CD20 monoclonal antibody(IDEC-C2B8) in patients with recurrent B-cell lymphoma. Blood.1994;84:2457-2466

2. Cheson B D. Rituximab: clinical development and future directions.Expert Opin Biol Ther. 2002;2:97-110

3. Alas S, Emmanouilides C, Bonavida B. Inhibition of interleukin 10 byRituximab results in Down-regulation of Bcl-2 and sensitization ofB-cell Non-Hodgkin's lymphoma to apoptosis. Clin Cancer Res.2001;7:709-723

4. Chow K U, Sommerlad W D, Boehrer S, Schneider B, Seipelt G, Rummel MJ, Hoelzer D, Mitrou P S, Weidmann E. Anti-CD20 antibody (IDEC-C2B8,rituximab) enhances efficacy of cytotoxic drugs on neoplasticlymphocytes in vitro: role of cytokines, complement, and caspases.Haematologica. 2002;87:33-43

5. Reff M E, Carner K, Chambers K S, Chinn P C, Leonard J E, Raab R,Newman R A, Hanna N, Anderson D R. Depletion of B cells in vivo by achimeric mouse human monoclonal antibody to CD20. Blood. 1994;83:435-445

6. McLaughlin P, Grillo-Lopez A J, Kink B K, Levy R, Czuczman M S,Williams M E, Heyman M R, Bence-Bruckler I, White C A, Cabanillas F,Jain V, Ho A D, Lister J, Wey K, Shen D, Dallaire B K. Rituximabchimeric anti-CD20 monoclonal antibody therapy for relapsed indolentlymphoma: half of patients respond to four-dose treatment program. JClin Oncol. 1998;16:2825-2833

7. Davis T A, Grillo-Lopez A J, White C A, McLaughlin P, Czuczman M S,Link B K, Maloney D G, Weaver R L, Rosenberg J, Levy R. Rituximabanti-CD20 monoclonal antibody therapy in non-Hodgkin's lymphoma: safetyand efficacy of re-treatment. J Clin Oncol. 2000;18:3135-3143

8. Davis T A, Czerwinski D K, Levy R. Therapy of B-cell lymphoma withanti-CD20 antibodies can result in the loss of CD20 antigen expression.Clin Cancer Res. 1999;5:611-615

9. Cartron G, Dacheux L, Salles G, Solal-Celigny P, Bardos P, ColombatP, Watier H. Therapeutic activity of humanized anti-CD20 monoclonalantibody and polymorphism in IgG Fc receptor FcgammaRIIIa gene. Blood.2002;99:754-758

10. Golay J, Zaffaroni L, Vaccari T, Lazzari M, Borleri G M, BernasconiS, Tedesco F, Rambaldi A, Introna M. Biologic response of B lymphomacells to anti-CD20 monoclonal antibody rituximab in vitro: CD55 and CD59regulate complement-mediated cell lysis. Blood. 2000;95:3900-3908

11. Bohen S P, Troyanskaya O G, Alter O, Warnke R, Botstein D, Brown PO, Levy R. Variation in gene expression patterns in follicular lymphomaand the response to rituximab. Proc Natl Acad Sci USA.2003;100:1926-1930

12. Bohn J A, BeMiller J N. (1-3)-B-D-Glucans as biological responsemodifiers:a review of structure-functional activity relationships.Carbohydr Polymers. 1995;28:3-14

13. Ross G D, Cain J A, Myones E L, Newman S L, Lachmann P J.Specificity of membrane complement receptor type three (CR3) forbeta-glucans. Complement Inflamm. 1987;4:61-74

14. Xia Y, Vetvicka V, Yan J, Hanikyrova M, Mayadas T, Ross G D. Thebeta-glucan-binding lectin site of mouse CR3 (CD11b/CD18) and itsfunction in generating a primed state of the receptor that mediatescytotoxic activation in response to iC3b-opsonized target cells, JImmunol. 1999;162:2281-2290

15. Cheung N K, Modak S. Oral (1-3),(1-4)-beta-glucan syngergizes withanti-ganglioside GD2 monoclonal antibody 3F8 in the therapy ofneuroblastoma. Clin Cancer Res. 2002;8:1217-1223

16. Cheung N K, Modak S, Vickers A, Knuckles B. Orally administeredbeta-glucans enhance anti-tumor effects of monoclonal antibodies. CancerImmunol Immunother. 2002;51:557-564

17. Koehne G, Gallardo H F, Sadelain M, O'Reilly R J. Rapid selection ofantigen-specific T lymphocytes by retroviral transduction. Blood.2000;96:109-117

18. Wei B R, Ghetie M A, Vitetta E S. The combined use of an immunotoxinand a radioimmunoconjugate to treat disseminated human B-cell lymphomain immunodef icient mice. Clin Cancer Res. 2000;6:631-642

19. Vardi Y, Ying Z, Zhang C-H. Two-sample tests for growth curves underdependent right censoring. Biometrika. 2001;88:949-960

20. Yan J, Vetvicka V, Xia Y, Coxon A, Carroll M C, Mayadas T N, Ross GD. B-glucan a “Specific” biologic response modifier that uses antibodiesto target tumors for cytotoxic recognition by leukocyte complementreceptor type 3 (CD11/CD18). J Immunol. 1999;163:3045-3052

21. Treon S P, Mitsiades C, Mitsiades N, Young G, Doss D, Schlossman R,Anderson K C. Tumor cell expression of CD59 is associated withresistance to CD20 serotherapy in patients with B-cell malignancies. JImmunother. 2001;24:263-271

22. Jurianz K, Ziegler S, Garcia-Schuler H, Kraus S, Bohana-Kashtan O,Fishelson Z, Kirschfink M. Complement resistance of tumor cells: basaland induced mechanisms. Mol Immunol. 1999;36:929-939

23. Vetvicka V, Thornton B P, Wieman T J, Ross G D. Targeting of naturalkiller cells to mammary carcinoma via naturally occurring tumorcell-bound iC3b and beta-glucan-primed CR3 (CD11b/CD18). J Immunol.1997;159:599-605

24. Adachi Y, Okazaki M, Ohno N, Yadomae T. Enhancement of cytokineproduction by macrophages stimulated with (1→3)-beta-D-glucan, grifolan(GRN), isolated from Grifola frondosa. Biol Pharm Bull.1994;17:1554-1560

25. Van der Kolk L E, Grillo-Lopez A J, Baars J W, Hack C E, van Oers MH. Complement activation plays a key role in the side-effects ofrituximab treatment. Br J Haematol. 2001;115:807-811

EXAMPLE III

Barley β-Glucan Extract Synergizes with IgM Antibodies

Natural IgM antibody from human serum when administered i.v. wascytotoxic for human neuroblastoma (NB) cells effecting growth arrest ofsubcutaneous solid human NB xenografts in nude rats. (1, 2) IgM wastaken up by the tumors with massive perivascular complement activationand accumulation of granulocytes after 24 hours. (3) In metastatic NBmodel, IgM antibody was effective in eliminating tumors in 90% of themice.(4) The absence of this anti-NB IgM antibody during infancy andamong NB patients (of any age), and its prevalence after 12 months ofage has raised the hypothesis that natural IgM antibodies could play arole as an immunological control mechanism against NB.(S) 3G6 is ananti-GD2 mouse IgM monoclonal antibody (MoAb). Within 48 hours afteri.v. injection of biotinylated 3G6, subcutaneous NB xenografts showedmembrane staining of tumor cells. Although 3G6 had lower meanfluorescence (53±19 fluorescent channel units, n=7 mice) when comparedto 3F8, an IgG MoAb (149±44, n=7), 3G6 plus beta-glucan was effectiveagainst sc human NB (p<0.05), with a dose response curve (FIG. 4)comparable to that of 3F8. (6) These findings were consistent with thoseusing human natural anti-NB IgM. (1, 2) These data support the idea thatbeta-glucan can enhance not just IgG inducing vaccines, but also IgMinducing vaccines.

REFERENCES OF EXAMPLE III

1. David K, Ollert M W, Juhl H, et al: Growth arrest of solid humanneuroblastoma xenografts in nude rats by natural IgM from healthyhumans. Nat Med 2:686-9, 1996

2. Ollert M W, David K, Schmitt C, et al: Normal human serum contains anatural IgM antibody cytotoxic for human neuroblastoma cells. Proc NatlAcad Sci USA 93:4498-503, 1996

3. Ollert M W, David K, Vollmert C, et al: Mechanisms of in vivoanti-neuroblastoma activity of human natural IgM. Eur J Cancer33:1942-8, 1997

4. Engler S, Thiel C, Forster K, et al: A novel metastatic animal modelreflecting the clinical appearance of human neuroblastoma: growth arrestof orthotopic tumors by natural, cytotoxic human immunoglobulin Mantibodies. Cancer Res 61:2968-73, 2001

5. Erttmann R, Schmitt C, Ollert M W, et al: Naturally occurring humoralcytotoxicity against neuroblastoma (NB) cells in healthy persons and NBpatients. Pediatr Hematol Oncol 13:545-8, 1996

6. Cheung N, Modak S: Oral (1-3),(1-4)-beta-glucan syngergizes withanti-ganglioside GD2 monoclonal antibody 3F8 in the therapy ofneuroblastoma. Clin Cancer Res 8:1217-1223, 2002

EXAMPLE IV

(1→3),(1→6) β-Glucan Derived from Baker's Yeast (Derived fromSaccharomyces cerevisiae) is also Effective in Enhancing AntibodyTherapy of Cancer

LAN-1 tumor cells were planted (2×10⁶ cells) in 100 μl of Matrigel(Sigma) subcutaneously. Tumor dimensions were measured two to threetimes a week with vernier calipers, and tumor size was calculated as theproduct of the two largest perpendicular diameters. All treatmentstudies started in groups of 4-5 mice when tumor diameters reached 0.7to 0.8 cm. Mice received antibody (3F8 or 3G6) treatment (200 ug perday) i.v. (by tail vein injection) twice weekly×5 doses and oralbeta-glucan (400 ug per day) by intragastric injection every day for atotal 14-18 days. (See FIG. 5 and 6)

Glucans derived from cell walls of yeasts, such as Saccharomycescervisiae or mutant yeast strains described in U.S. Pat. No. 5,250,436,the disclosure of which is incorporated herein in its entirety byreference, may be used in the above compositions. Glucans having β(1-3)and β(1-6) linkages may be prepared by the process described in U.S.Pat. Nos. 5,233,491 and 4,810,646, the disclosures of which areincorporated herein in their entirety by reference. Soluble or aqueousglucans which are suitable for oral administration may be produced bythe process described in U.S. Pat. Nos. 4,810,646 and 5,519,009, thedisclosures of which are incorporated herein in their entirety byreference. Beta-glucans such as the Soluble beta-1,3/1,6 glucan or SBGmanufactured by Biotec Pharmacon (Norway) may also be used.

In similar experiments a subcutaneous lymphoma model was studied. Here5×10⁶ cells suspended in 0.1 ml of Matrigel (Becton-Dickinson, FranklinLakes, N.J.) were planted into mice flanks. Tumor dimensions weremeasured two to three times a week and tumor size was calculated asproduct of the two largest diameters. Mice were sacrificed when maximumtumor dimension exceeded 20 mm. 200 μg rituximab (Genentech, SanFrancisco, Calif.) was injected intravenously twice weekly for a totalof eight injections and 400 μg glucan administered orally viaintragastric gavage daily for 29 days. Mice were weighed weekly andobserved clinically at least once daily. The rate of tumor response andthe percent of mice achieving complete remissions were comparablebetween barley glucan and yeast glucan. These series of subcutaneoustumor models showed that soluble yeast (1→3),(1→6) beta-glucan of largemolecular weight (>10,000 Daltons) is equally potent as barley(1→3),(1→4) beta-glucan. In addition, the source and physical form ofyeast glucan can make substantial differences.

Metastatic lymphoma model was also studied. A model of disseminatedtumors was established in SCID mice as previously described. (1)Briefly, 5×10⁶ Daudi cells in 100 μl normal saline were injectedintravenously (i.v.) into SCID mice. Tumors grew systemically and micebecame paralyzed when tumor cells infiltrated the spinal canal,resulting in hind-leg paralysis. Mice were sacrificed at onset ofparalysis or when animals lost 10% of their body weight. Therapy wasinitiated ten days after injection of tumor cells. 40 μg rituximab(Genentech, San Francisco, Calif.) was injected intravenously twiceweekly for a total of eight injections and 400 μg glucan administeredorally via intragastric gavage daily for 29 days. Mice were weighedweekly and observed clinically at least once daily. (See FIG. 7)

Again both barley glucan and yeast glucan showed comparable effect whencombined with Rituxan. Neither barely glucan nor yeast glucan has anyeffect on survival when used alone (data not shown).

REFERENCES FOR EXAMPLE IV

1. Wei B R, Ghetie M A, Vitetta E S: The combined use of an immunotoxinand a radioimmunoconjugate to treat disseminated human B-cell lymphomain immunodeficient mice. Clin Cancer Res 6:631-642, 2000

EXAMPLE V

Mechanism by which Orally Administered β-Glucans Function withAnti-Tumor Monoclonal Antibodies to Mediate Tumor Regression. (1)

Using syngeneic tumor (GD2+RMA-S) in wild type (WT) C57B1/6 mice versuseither CR3-deficient (CD11b −/−) or C3-deficient (C3 −/−) C57B1/6 mice,MoAb alone elicited no tumor regression, whereas combining the i.v.anti-GD2 MoAb with oral barley or yeast beta-glucan elicited significantregression in WT but not in CR3-deficient mice. Moreover, the combinedtreatment with i.v. MoAb and oral beta-glucans produced 60-100%tumor-free survivors in WT mice, but only 0-20% survival in theCR3-deficient mice. These experiments demonstrated a near absoluterequirement for leukocyte CR3 for the anti-tumor effect, especially whenoral barley beta-glucan was given with anti-tumor MoAb. A therapyprotocol comparing WT to C3-deficient mice similarly showed that oralbeta-glucan therapy required serum C3. When barley beta-glucan and yeastbeta-glucan were labeled with fluorescein (BG-F and YG-F) and given tomice by intragastric injection, the trafficking of beta-glucan wasfollowed. Within three days of daily oral administration of BG-F orYG-F, macrophages in the spleen and lymph nodes containedfluorescein-labeled beta-glucan. After 4 d, YG-F and BG-F were alsoobserved in macrophages in bone marrow. When the uptake of YG-F and BG-Fby WT versus CR3-deficient mice was compared, no differences wereapparent in either the percentage of macrophages containing ingestedbeta-glucan-F or the amount of beta-glucan-F per cell. Thus, the uptakeof barley and yeast beta-glucan by gastrointestinal macrophages does notrequire CR3 and is likely mediated instead by Dectin-1.(2) Macrophagesin vitro and in the marrow were able to degrade large molecules ofbarley or yeast beta-glucan into smaller biologically-active fragmentsof beta-glucan that are then released.

To determine if the soluble beta-glucan-F released by macrophages hadindeed been taken up by bone marrow granulocytes, groups of WT orCR3-deficient mice that had been given YG-F or BG-F for 10 days wereinjected i.p. with thioglycolate medium to elicit the marginated pool ofbone marrow granulocytes into the peritoneal cavity. Only WTgranulocytes were able to pick up the YG-F and BG-F released frommacrophages. These data suggest a sequential ingestion of beta-glucan bygastrointestinal macrophages that shuttle the beta-glucan to the bonemarrow where soluble degradation fragments are released and taken up bygranulocytes via membrane CR3. When peritoneal granulocytes wereisolated from WT and CR3-deficient mice that had been given oralbeta-glucan, only WT granulocytes were able to kill iC3b-coated tumorcells in vitro. These experiments show that bone marrow granulocytes andtissue macrophages acquire membrane CR3-bound soluble beta-glucan fromgastrointestinal macrophages, and that this bound beta-glucan primes theCR3 of both granulocytes and macrophages so that when they are recruitedto a site of inflammation they are able to kill iC3b-coated tumor cells.

REFERENCES FOR EXAMPLE V

1. Hong F, Yan J, Baran J T, et al: Mechanism by which orallyadministered beta(1,3)-glucans function with anti-tumor monoclonalantibodies to mediate tumor regression and tumor-free survival. J ExpMed, 2004

2. Herre J, Gordon S, Brown G D: Dectin-1 and its role in therecognition of beta-glucans by macrophages. Mol Immunol 40:869-76, 2004

EXAMPLE VI

Soluble β-Glucan can be Used as a Conduit for Plasmids.

The major obstacles for the delivery of DNA, RNA and proteins orally arethe acidic and proteolytic environment of the stomach, and limiteduptake of proteins by the GALT. It is believed that M cells within thePeyer's patches and phagocytes are the predominant vehicles for uptakeof microparticulates. However, nanoparticles may also access GALT via aparacellular mechanism1,2 and by transcytosis.³ In either case, particleuptake observed can be improved using particles with mucoadhesiveproperties or affinity for receptors on cells. Many polymers have beenused to fabricate nanoparticles are mucoadhesive. Among them arealginate, carrageenans, and pectin. Although these materials were oftenused as the core polymers in nanoparticulates, no specific receptor hasbeen identified for these polymers and the efficiency of uptake remainssuboptimal. Dectin-1 is now known to be a universal receptor forβ-glucan, and is found in many human tissues including monocytes andphagocytes. The gelling properties of high molecular weight β-glucanallows RNA, DNA and proteins to be embedded. Since sugars are highlyresistant to acid conditions and enzymes, proteins, RNA and DNA remainprotected during their passage through the gastrointestinal tract.Through the high affinity Dectin-1 receptor for β-glucan, thesesubstances can be introduced into the phagocytes as potential vehiclesto the rest of the body.

The peGP-C1 vector (See FIG. 8) was purchased from BD Biosciences (PaloAlto, Calif.) and prepared according to manufacturers' instructions.pEGFP-C1 encodes a red-shifted variant of wild-type GFP (1-3) which hasbeen optimized for brighter fluorescence and higher expression inmammalian cells. (Excitation maximum=488 nm; emission maximum=507 nm.)The vector backbone also contains an SV40 origin for replication inmammalian cells only if they express the SV40 T-antigen. A bacterialpromoter upstream of this cassette expresses kanamycin resistance in E.coli. The pEGFP-C1 backbone also provides a pUC 30 origin of replicationfor propagation in E. coli and an f1 origin for single-stranded DNAproduction.

Mice were fed with 50 μg pEGFP-c1 plasmid mixed into 400 μg beta-glucan(˜200,000 Daltons) in 100 μl saline by oral gavage while control micewere given plasmid alone. Oral feeding was done for 3 consecutive days(days 1, 2 and 3). So 50 μl blood taken from tail vein were analysed byFCAS analysis after lysis of RBC and the % of GFP-expressing cells inthe monocyte population were recorded. The mean ratio of % green cellsin glucan verus no glucan groups (n=4-9 mice per group) is presented inFIG. 9. Thoughout the 14 days of the experiment, % green monocytes inthe no-glucan group remained stable at background levels. On the otherhand, after day 1 of oral gavage, there was a consistent higher % ofcirculating green monocytes, which peaked around day 8. Since the GFP isnot normally found in mouse monocytes, the presence of green cells isconsistent with GFP protein expression following entry of the plasmidinto the monocytes which circulate in the blood.

The experiment was repeated using barley β-glucan of higher molecularweight (˜350,000 Daltons) with better gelling properties. In FIG. 10,similar kinetics was seen, with a higher percent of green cells thatpersisted from day 8 through day 11 (n=4 mice per group).

Presence of GFP mRNA was tested using quantitative reverse-transcriptionPCR analysis. Mice were fed with 50 μg pEGFP-c1 plasmid mixed into 400μg high molecular weight (˜350,000 Daltons) beta-glucan in 100 μl salineby oral gavage while control mice were given plasmid alone. 50 μlperipheral blood was used to extract total RNA, reverse transcribed andquantitative real-time PCR was performed using a modification of themethod previously described.⁴ The house keeping gene mouse GAPDH is usedas internal control. Transcript level is calculated using a known GFPand GAPDH standard. Transcript units are calculated separately for GFPand GAPDH and results as a ratio of GFP over GAPDH. In FIG. 11, the meanRNA level (GFP/GAPDH) is expressed as a ratio of glucan versus no glucangroups (n=4 mice per group). GFP mRNA was detected up to day 10.

REFERENCES FOR EXAMPLE VI

1. Damge C, Aprahamian M, Marchais H, et al: Intestinal absorption ofPLAGA microspheres in the rat. J Anat 189 ( Pt 3):491-501, 1996

2. Jani P, Halbert G W, Langridge J, et al: Nanoparticle uptake by therat gastrointestinal mucosa: quantitation and particle size dependency.J Pharm Pharmacol 42:821-6, 1990

3. Florence A T: The oral absorption of micro- and nanoparticulates:neither exceptional nor unusual. Pharm Res 14:259-66, 1997

4. Cheung I Y, Lo Piccolo M S, Collins N, et al: Quantitation of GD2synthase mRNA by real-time reverse transcription-polymerase chainreaction: utility in bone marrow purging of neuroblastoma by anti-GD2antibody 3F8. Cancer 94:3042-8, 2002

1. A composition for oral uptake of substance comprising an appropriateamount of carbohydrates.
 2. A composition for the oral delivery of oneor more substances comprising an effective amount of an orallyadministered beta-glucan and one or more chemotherapeutic agents.
 3. Thecomposition of any one of claims 1 or 2, wherein the carbohydrate isglucan.
 4. The composition of claim 3, wherein the glucan contains1,3-1,6 or 1,3-1,4 mixed linkages or a mixture of both 1,3-1,6 and1,3-1,4 mixed linkages.
 5. The composition of claim 3, wherein theglucan enhances the efficacy of chemotherapeutic agents or anti-cancerantibodies.
 6. The composition of claim 3, wherein the glucan is derivedfrom grass, plants, mushroom, yeast, barley, fungi, wheat or seaweed. 7.The composition of claim 3, wherein the glucan is of high molecularweight.
 8. The composition of claim 3, wherein the substance is apeptide, protein, RNA, DNA or plasmid.
 9. The composition of claim 3,wherein the substance is a chemotherapeutic agent.
 10. A compositioncomprising an effective amount of orally administered (1→3),(1→6) or(1→3),(1→4) beta-glucan capable of enhancing efficacy of IgM antibodies.11. The composition of claim 10, wherein the antibody is an antibodyagainst cancer.
 12. The composition of claim 11, wherein the antibody isa tumor-binding antibody.
 13. The composition of claim 12, wherein theantibody is capable of activating complement.